Development of Sensitive and Simultaneous Determination Method for Thirty-Seven d/l-Amino Acids by Automatic Pre-column o-Phthalaldehyde- Derivatization with Chiral Thiol using High Performance Liquid Chromatography

A complementary HPLC method for the determination of 37 proteinogenic d/l-amino acids excluding proline by analyzing each sample twice while automatically switching between the two separation methods using two chiral thiols of N-acetyl-l-cysteine (NAC) and N-isobutyryl-l-cysteine (NIBC) was previously reported. In this study, separation of o-phthalaldehyde (OPA)/NIBC-derivatized proteinogenic d/l-amino acids with higher hydrophobicity under a single analytical method was investigated. By increasing the methanol ratio of the mobile phase and setting the column temperature to 20 oC, it was possible to separate the 37 fluorescent diastereomers of the proteinogenic d/l-amino acids in a single analysis within 66 minutes, with an analysis cycle of 87 minutes. The peak area repeatability for each diastereomer was 1.6% RSD or less because of automating the OPA/NIBC derivatization of proteinogenic d/l-amino acids. The calibration curve linearity was 0.999 or greater for each diastereomer. As a result of applying this method to liquor samples, it was found that the overall ratio of d-amino acid to d/l-amino acid was 6% or less, but it was quantifiable. Principal component analysis of the five liquor samples showed that these samples were classified by type of liquor. The established method has the potential to be applied to liquor profiling.